Polymyxin is a complex of very similar polypeptide antibiotics isolated from different types of Bacillus polymyxa strains and related species. It is a cyclic polypeptide with free amino acid groups containing characteristic constituents such as α,γ-aminobutanic acid, L-treonine and fatty acids (6-methyloktanoyl acid, 3-hydroxy-6-methyloktanoyl acid, 6-methylheptanoyl acid, heptanoyl acid, oktanoyl acid, nonanoyl acid). This complex was divided by semi-preparative PLRP-S with reverse phase on 7 components (Orwa J. A., Govaert C., Busson R., et all, 2001, J. Chromatography A, 912, 369-373). The structure was characterized by 1H- and 13C-NMR methods and molecular weight was measured by mass spectroscopy method. It confirmed the structure of polymyxin B1, Ile-B1, B2, B3 and B4. The further two components, polymyxin B5 and B6, were isolated for the first time and characterized at this study.
Polymyxin B and E have lower toxicity like another polymyxin and they are preferred for medical use, mainly antibacterial more effective polymyxin B.
The procedures for recovery of polymyxin from fermentation broth in principal are based on application of activated carbon and ion-exchange. U.S. Pat. No. 2,565,057, describes a character of polypeptide antibiotic base as a metabolic products of Bacillus aerosporus and Bacillus polymyxa; there are listed also methods of its recovery from culture media by using adsorption properties of activated carbon. The first part of impurities are separated from the filtrate of the broth by activated carbon in acid conditions (pH 2.5). The discolored solution of antibiotic is adjusted to neutral pH and then it is adsorbed onto active carbon. The adsorbed antibiotic is eluted by aqueous solution of acid acetone. The eluate is purified by adjustment of pH, separation of acetone and re-adsorption on activated carbon, neutralization of the eluate with CaCO3 and finalization by lyophilization of the antibiotic solution. Another alternative of antibiotic purification from eluate is preparation of insoluble salts such as precipitation by picric acid and reconversion of this salt into aqueous soluble salts usually in a form of sulphates (M. Harold et al.: Antibiotika CsAV Praha, 1957, page 285-288).
In a further two patents (GB 742 589 and GB 782 926) the filtrates from the fermentation broth are purified in weak acid ion-exchangers in different column systems. The adsorbed polymyxin is eluted by a solution of mineral acid or buffer solution with a pH in a range from 3 to 5. The obtained eluate is purified on an anion exchanger or using a strong acid cation exchanger. This repurified eluate is concentrated and polymyxin is precipitated by addition of alkaline solution. Precipitation occurs in a range of temperature from 60° C. to 90° C.
U.S. Pat. No. 3,132,994 (1964) describes the method of increased purification of crude sulphates- and N-methylsulphonates of polymyxin B and E. The background of this method is oxidation of these salts solutions in acid or neutral pH (pH 3.5 till 7.5) with 1% water solution of KMnO4. In the next step the low molecular weight materials are separated, including Mn-cations in a strong acid cation exchanger in H+ cycle, where the polymyxin is not adsorbed.
The procedure for recovery and purification of amphoteric and alkaline antibiotics including polymyxin B, using reactive extraction into organic solvent limited with aqueous mixture and extraction in a present of carriers di-(2-ethylhexyl)-ester or dinonyl ester of phosphoric acid is described in patent GB 979 887 (1964). Polymyxin B is extracted from the broth into 1% solution of di-(2-ethylhexyl)-ester of phosphoric acid in buthylacetate at pH 7. Purification of polymyxins B and E from acid solutions by alkaline reagent in a present of chelating agents to form complexes of cations Ca, Mg, Mn, Fe is describe in patents GB 1089765 (1964) and U.S. Pat. No. 3,413,398. The antibiotic are precipitate in a form of base solution by using alkaline agents such as hydroxides and carbonates of alkaline metals and ammonium hydroxide at pH 8.5 till 11 and addition of chelating agents avoid coprecipitation of impurities. There is described a list of chelating agents, which were added in equivalent ratio as the polyvalent inorganic ions present. The precipitation of antibiotic base may occur in 10% water solution of acetone. In the examples of these patents are described methods for purification of polymyxin B, where the acid solution of crude polymyxin B is oxidized with KMnO4, according to the patent GB 991602. The leftover KMnO4 is eliminated by H2O2. This is continued further with precipitation of base in presence of ethylenediamine-N,N,N,N′ tetraacetic acid tetra sodium salt. The base is filtered off, washed and vacuum-dried. The base is converted to the sulphate of polymyxin B by molar equivalent of H2SO4. In described procedures of recovery of polymyxin B solid substance from broth, the broth is purified by complicated methods and crude antibiotic is obtained. The crude antibiotic must be purified in several steps to get a pure substance.